I am trying to understand the immunoprecipitation as I need to perform a SILAC based mass spectrometry experiment. Basically I have grown my cells in two conditions (light and heavy media). Now after extracting the proteins I need to check the levels of just four proteins in my sample via SILAC. I know these proteins are low abundant. So I will be immunoprecipitating these proteins from the two conditions. In the papers I read online, people do a western blot after immunoprecipitation. I did not get the reason for doing this. I mean can I directly subject the immunoprecipitated sample to trypsin digestion, then mix them in equal amount and do a SILAC?
Western blot is mainly to confirm immunoprecipitation. How efficient it was (check the unbound fraction), how clean (check for proteins that should not co-IP), whether it worked at all (check the presence of the bait). Also it should be equal efficiency in all samples that you are going to compare: what if one of the samples got less resin, or for some reason did not bind properly.
Thanks Maria, so if I have a highly specific monoclonal antibody for my protein of interest, then I can skip WB, right??
Sarvesh, the WB is not necessary to obtain results from the SILAC-MS approach, but rather confirmation by a second, non-MS-based method which is desirable/required if you want to use these results for a biochemical research project and publication. As it is also faster to do (usually) than MS, people usually do WB first and if they are confident their protein is in the pulldown, they then do MS.
Regarding IP - if your proteins of interest are low abundance then yes, IP is highly recommended as an enrichment step for both the SILAC-MS and the WB approach. Especially for MS from lysed cells, detection of low abundance proteins by MS can be like rolling dice.
You can always do WB first to check whether your antibody is working or not? If it does, then go for MS analysis...
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